Peroxisome proliferator activated receptor ligand and process for producing the same

ABSTRACT

The present invention easily and efficiently provides a peroxisome proliferator-activated receptor ligand, and a composition for amelioration of insulin resistance or for prevention and/or amelioration of the insulin resistance syndrome containing the same, as an active ingredient. 
     The present invention relates to a peroxisome proliferator-activated receptor ligand which comprises a prenylflavonoid, a chalcone derivative exclusive of prenylflavonoids, a flavonol derivative exclusive of prenylflavonoids, and a salt, a glycoside and/or an esterified substance thereof acceptable as a pharmaceutical preparation or a food or a beverage; a composition containing the above ligand; a plant-derived extract containing the above ligand; and a process for producing the above extract.

TECHNICAL FIELD

The present invention relates to a peroxisome proliferator-activatedreceptor ligand, a process for producing the same, and a composition foramelioration of insulin resistance or for prevention and/or ameliorationof the insulin resistance syndrome (type 2 diabetes, hyperinsulinemia,dyslipidemia, obesity, hypertension and arteriosclerotic disease)containing the peroxisome proliferator-activated receptor ligand as anactive ingredient.

BACKGROUND ART

Peroxisome proliferator-activated receptor (PPAR) is a ligand-dependenttranscription regulator belonging to an intranuclear receptor family.This family is identified as a family of transcription regulatorsinvolved in regulating expression of a lipid metabolism-maintaining genegroup. In mammalian animals, the presence of 3 subtypes, i.e. PPARα,PPARδ (PPARβ, NUC-1, FAAR) and PPARγ, is known, and PPARα (is expressedmainly in the liver and PPARδ is universally expressed. For PPARγ, thereare 2 isoforms, i.e. PPARγ1 and PPARγ2, and PPARγ1 is expressed not onlyin an adipose tissue but also in an immune organ, adrenal gland andsmall intestine. PPARγ2 is expressed specifically in an adipose tissue,and is a master regulator which regulates differentiation and maturationof an adipocyte (Teruo Kawada, “Igaku No Ayumi”, 184, 519-523, 1998).

As PPARγ ligands, synthetic compounds including thiazolidine derivativessuch as troglitazone, pioglitazone and rosiglitazone are known. As PPARγligands, naturally occurring compounds including arachidonic acidmetabolites such as 15-deoxy-Δ^(12,14)-prostaglandin J₂ andΔ¹²-prostaglandin J₂, unsaturated fatty acids such as ω-3 multivalentunsaturated fatty acid, α-linolenic acid, eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA), and eicosanoids such as9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid areknown (J. Auwerx, Diabetologia, 42, 1033-1049, 1999). Japanese KokaiPublication 2000-355538 discloses a C₁₀₋₂₆ conjugated unsaturated fattyacid having a conjugated triene structure or a conjugated tetraenestructure. For flavonoids, chrysin and apigenin as flavone derivativesand kaempferol as a flavonol derivative are reported to be PPARγ ligands(Y. C. Liang, et al., FEBS Letters, 496, 12-18, 2001). A flavonoid is acomponent contained widely in plants, and is known to have antioxidantproperties. However, it is not known that a prenylflavonoid containedonly in a specific plant is a PPARγ ligand.

The agonistic activity of a thiazolidine derivative as PPARγ ligand iscorrelated with its hypoglycemic action, thus the derivative hasattracted attention for its relationship with an ameliorating action oninsulin resistance and been developed as an insulin resistanceameliorator for type 2 diabetes mellitus (non-insulin dependent diabetesmellitus: NIDDM). That is, a thiazolidine derivative, which is a PPARγligand, activates PPARγ thereby increasing a normally functioning smalladipocyte differentiated from a preadipocyte. Thereafter, a hypertrophicadipocyte, in which over-production and over-secretion of TNFα and afree fatty acid inducing insulin resistance, is reduced with apotosis.As a result, insulin resistance is ameliorated (A. Okuno, et al.,Journal of Clinical Investigation, 101, 1354-1361, 1998). A PPARγ ligandameliorates insulin resistance and is thus also effective in preventingand/or ameliorating the insulin resistance syndrome including not onlytype 2 diabetes mellitus but also hyperinsulinemia, dyslipidemia,obesity (in particular visceral fat obesity), hypertension andarteriosclerosis (R. A. DeFronzo & E. Ferrannini, Diabetes Care, 14,173-194, 1991). Pathologic conditions in the same category as theinsulin resistance syndrome include Syndrome X (G. M. Reaven, Diabetes,37, 1595-1607, 1988), the deadly quartet (N. M. Kaplan, Archives ofInternal Medicine, 149, 1514-1520, 1989) and the visceral fat syndrome(Y. Matsuzawa, Diabetes/Metabolism Reviews, 13, 3-13, 1997).

A PPARγ ligand is also effective in preventing and/or amelioratinginflammations and cancers because of its inhibition of inflammatorycytokine production (C. Jiang, et al., Nature, 391, 82-86, 1998) and itsinduction of apotosis thereby inhibiting growth of cancer cells (Y.Tsubouchi, et al., Biochemical and Biophysical Research Communications,270, 400-405, 2000).

SUMMARY OF THE INVENTION

In view of the foregoing, a PPARγ ligand has an effect on ameliorationof insulin resistance, and on prevention and/or amelioration of theinsulin resistance syndrome such as type 2 diabetes mellitus,hyperinsulinemia, dyslipidemia, obesity (in particular visceral fatobesity), hypertension and arteriosclerosis. The object of the presentinvention is to provide a PPAR ligand found in naturally occurringmaterials, a process for easily and efficiently producing the same, anda composition for amelioration of insulin resistance or for preventionand/or amelioration of the insulin resistance syndrome containing theligand as an active ingredient.

The present inventors searched for a substance having a PPARγ ligandactivity from eatable naturally occurring materials, and as a result,they found that an extract of plant such as licorice has a PPARγ ligandactivity. The present inventors intensively studied its activeingredient, and as a result they found that a specific component in theextract has a PPARγ ligand activity. Furthermore, the present inventorsfound that an organic solvent, preferably a fatty acid ester or awater-soluble organic solvent, particularly a water-soluble organicsolvent, can be used to efficiently extract such a component fromlicorice, to complete the present invention.

That is, the first aspect of the invention is concerned with

a peroxisome proliferator-activated receptor ligand

which comprises a prenylflavonoid, a chalcone derivative exclusive ofprenylflavonoids, a flavonol derivative exclusive of prenylflavonoids,and a salt, a glycoside and/or an esterified substance thereofacceptable as a pharmaceutical preparation or a food or a beverage.

The second aspect of the invention is concerned with

a plant-derived extract

which comprises a peroxisome proliferator-activated receptor ligandcomprising a prenylflavonoid, a chalcone derivative exclusive ofprenylflavonoids, a flavonol derivative exclusive of prenylflavonoids,and a salt, a glycoside and/or an esterified substance thereofacceptable as a pharmaceutical preparation or a food or a beverage.

The third aspect of the invention is concerned with

a composition for amelioration of insulin resistance or for preventionand/or amelioration of the insulin resistance syndrome

which comprises, as an active ingredient, a prenylflavonoid having anability to bind to a ligand-binding region of a peroxisomeproliferator-activated receptor, a chalcone derivative exclusive ofprenylflavonoids, a flavonol derivative exclusive of prenylflavonoids,and a salt, a glycoside and/or an esterified substance thereofacceptable as a pharmaceutical preparation or a food or a beverage.

The fourth aspect of the invention is concerned with

a composition for prevention and/or amelioration of inflammations andcancers

which comprises, as an active ingredient, a prenylflavonoid having anability to bind to a ligand-binding region of a peroxisomeproliferator-activated receptor, a chalcone derivative exclusive ofprenylflavonoids, a flavonol derivative exclusive of prenylflavonoids,and a salt, a glycoside and/or an esterified substance thereofacceptable as a pharmaceutical preparation or a food or a beverage.

The fifth aspect of the invention is concerned with

a process for producing the extract described above

which comprises extracting the extract from licorice having the ratio ofthe skin area to the whole surface area of 30% or more.

The sixth aspect of the invention is concerned with

a process for producing the extract described above

wherein the extraction from licorice is carried out with a fatty acidester or a water-soluble organic solvent.

The seventh aspect of the invention is concerned with

a process for producing the extract described above

which comprises extracting from licorice with an organic solvent havinga water content of 30% by volume or less.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, embodiments of the present invention are described indetail.

A PPAR ligand (preferably a PPARγ ligand) is a compound having PPARligand activity (preferably PPARγ ligand activity), that is, a compoundhaving an ability to bind to a ligand-binding region of PPAR (preferablyPPARγ). PPARγ ligand activity can be measured by, for example, areporter assay wherein a binding of the compound to a fusion protein ofa PPARγ ligand-binding region and GAL4 is evaluated in terms ofexpression of luciferase (B. M. Forman, et al., Cell, 83, 803-812,1995), competition binding assay using a protein having a PPARγligand-binding region (S. A. Kliewer, et al., Cell, 83, 813-819, 1995),etc. Generally, activity of a sample is compared with that of a solventcontrol in these assays, and a sample exhibiting higher activity thanthat of the solvent control and also recognized that the activity isdose-dependent is defined as the one having “PPARγ ligand activity.”

The PPAR ligand, preferably the PPARγ ligand, of the present inventionis at least one compound selected from the group consisting of aprenylflavonoid, a chalcone derivative exclusive of prenylflavonoids, aflavonol derivative exclusive of prenylflavonoids, and a salt, aglycoside and an esterified substance thereof acceptable as apharmaceutical preparation or a food or a beverage.

The salt acceptable as a pharmaceutical preparation or a food or abeverage includes, but is not limited to, sodium salts, potassium salts,magnesium salts, calcium salts, etc., for example.

The glycoside acceptable as a pharmaceutical preparation or a food or abeverage includes, but is not limited to, glycosides of monosaccharidessuch as glucose and galactose, glycosides of disaccharides oroligosaccharides, etc., for example.

The esterified substance acceptable as a pharmaceutical preparation or afood or a beverage includes, but is not limited to, acetates,propionates, phosphates, sulfates, etc., for example.

The composition of the present invention comprising, as an activeingredient, at least one species selected from these compoundsameliorates insulin resistance, and is thus useful as an agent forprevention and/or amelioration of the insulin resistance syndromeincluding diabetes mellitus.

The prenylflavonoid, which is the PPAR ligand of the present invention,is not particularly limited, but is preferably at least one compoundselected from the group consisting of a 3-arylcoumarin derivative, anisoflav-3-ene derivative, an isoflavan derivative, an isoflavanonederivative, an isoflavone derivative, a flavonol derivative, a flavanonederivative, a chalcone derivative and a dibenzoylmethane derivative.

The prenylflavonoid as referred herein is defined to be a compoundhaving at least one of the following (A) and (B) as a side chain offlavonoid: (A) a prenyl group, or (B) a structure having a 6-memberedring in which a prenyl group is bound to its adjacent hydroxyl group toform [—CH═CHC(CH₃)₂O—]. The term “flavonoid” is a generic term of agroup of substances having 2 phenyl groups bound each other via 3 carbonatoms, and examples thereof include flavone, flavonol, flavanone,flavanonol, isoflavone, isoflavonol, isoflavanone, isoflavan,isoflav-3-ene, 3-arylcoumarin, chalcone, dihydrochalcone,dibenzoylmethane, coumestan, pterocarpan, catechin, anthocyanidin, etc.

The 3-arylcoumarin derivative, which is a prenylflavonoid used in thepresent invention, is not particularly limited, but preferable examplesthereof include compounds represented by the following general formula(1). And for example, compounds shown in Table 1 such as glycycoumarin,glycyrin, gancaonin W, glyasperin L, kanzonol W, etc. may be mentioned.Derivatives thereof such as salts, glycosides and esterified substancesmay also be mentioned. Among these, glycycoumarin and glycyrin are morepreferable.

In the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 1 R1 R2 R3 R4 R5 R6 R7 3-Arylcoumarin(s) OCH₃ prenyl OH H OH H OHglycycoumarin OCH₃ prenyl OCH₃ H OH H OH glycyrin OCH₃ H OH H OH prenylOH gancaonin W OCH₃ H OH H OH —CH═CHC(CH₃)₂O— glyasperin L H H—OC(CH₃)₂CH═CH— OH H OH kanzonol W H H —OC(CH₃)₂CH═CH— OH H OCH₃ RL-P HH OH H —OC(CH₃)₂CH═CH— OH RL-U

The isoflav-3-ene derivative that is a prenylflavonoid used in thepresent invention is not particularly limited, but preferable examplesthereof include compounds represented by the following general formula(2). For example, compounds shown in Table 2 such as dehydroglyasperinC, dehydroglyasperin D, glabrene and RL-S. Derivatives thereof such assalts, glycosides and esterified substances may also be mentioned. Amongthese, dehydroglyasperin C, dehydroglyasperin D and glabrene are morepreferable. Dehydroglyasperin D is a novel compound found for the firsttime in the present invention.

In the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 2 R1 R2 R3 R4 R5 R6 R7 Isoflav-3-ene(s) OCH₃ prenyl OH H OH H OHdehydroglyasperin C OCH₃ prenyl OCH₃ H OH H OH dehydroglyasperin D H HOH H —OC(CH₃)₂CH═CH— OH glabrene H H —OC(CH₃)₂CH═CH— OH H OH RL-S

The isoflavan derivative that is a prenylflavonoid used in the presentinvention is not particularly limited, but preferable examples thereofinclude compounds represented by the following general formula (3). Forexample, compounds shown in Table 3 such as glyasperin C, glyasperin D,glyasperin I, licoricidin and licorisoflavan A. Derivatives thereof suchas salts, glycosides and esterified substances may also be mentioned.Among these, glyasperin D, glabridin, hispaglabridin B,4′-O-methylglabridin and 3′-hydroxy-4-O-methylglabridin are morepreferable.

In the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 3 R1 R2 R3 R4 R5 R6 R7 Isoflavan(s) OCH₃ prenyl OH H OH H OHglyasperin C OCH₃ prenyl OCH₃ H OH H OH glyasperin D OCH₃ prenyl OH HOCH₃ H OH glyasperin I OCH₃ prenyl OH H OH prenyl OH licoricidin OCH₃prenyl OCH₃ H OH prenyl OH licorisoflavan A OCH₃ prenyl OCH₃ H—OC(CH₃)₂CH═CH— OH kanzonol I H prenyl OH H —OC(CH₃)₂CH═CH— OHglyinflanin I H —CH═CHC(CH₃)₂O— H OH H OCH₃ gancaonin X OCH₃—CH═CHC(CH₃)₂O— H OH prenyl OCH₃ kanzonol H OCH₃ —CH═CHC(CH₃)₂O— H—OC(CH₃)₂CH═CH— OCH₃ kanzonol J H —CH═CHC(CH₃)₂O— H —OC(CH₃)₂CH═CH— Hglyinflanin J H H OH H OH prenyl OCH₃ gancaonin Z OCH₃ H OH H OH prenylOCH₃ kanzonol R H H OH prenyl OH prenyl OH kanzonol X H H—OC(CH₃)₂CH═CH— OH H OH glabridin H H —OC(CH₃)₂CH═CH— OH H OCH₃4′-O-methylglabridin H H —OC(CH₃)₂CH═CH— OH OH OCH₃ 3′-hydroxy-4′-O-methyl-glabridin H H —OC(CH₃)₂CH═CH— OH OCH₃ OH 3′-O-methylglabridin H H—OC(CH₃)₂CH═CH— OCH₃ OH OCH₃ glyasperin H H H —OC(CH₃)₂CH═CH— OH prenylOH hispaglabridin A H H —OC(CH₃)₂CH═CH— OH —CH═CHC(CH₃)₂O—hispaglabridin B H H —OC(CH₃)₂CH═CH— OCH₃ —CH═CHC(CH₃)₂O—methylhispaglabridin B H H —OC(CH₃)₂CH═CH— —OC(CH₃)₂CH═CH— H glyinflaninK H H OH H —OC(CH₃)₂CH═CH— OCH₃ gancaonin Y H H OH H OH —CH═CHC(CH₃)₂O—phaseollinisoflavan H H OH prenyl OH —CH═CHC(CH₃)₂O— 8-prenyl-phaseollinisoflavan

The isoflavan on derivative that is a prenylflavonoid used in thepresent invention is not particularly limited, but preferable examplesthereof include compounds represented by the following general formula(4). For example, compounds shown in Table 4 such as glyasperin B,glyasperin K, kanzonol G, 3′-prenyl-kievitone and glyasperin F.Derivatives thereof such as salts, glycosides and esterified substancesmay also be mentioned. Among these, glyasperin B andglycyrrhisoflavanone are more preferable.

In the formula, at least one of R1 to R8 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 4 R1 R2 R3 R4 R5 R6 R7 R8 Isoflavanone(s) OH prenyl OCH₃ H OH H OHH glyasperin B OH prenyl OCH₃ H OH H OCH₃ H glyasperin K OH prenyl OCH₃H OH prenyl OH H kanzonol G OH H OH prenyl OH prenyl OH H3′-prenyl-kievitone (3′,8-diprenyldalbergioidin) OH H OH H—OC(CH₃)₂CH═CH— OH H glyasperin F OH H OH prenyl —OC(CH₃)₂CH═CH— OH Hglyasperin J OCH₃ H OH H H OH —CH═CHC(CH₃)₂O— glycyrrhisoflavanone OCH₃H OH H OH —CH═CHC(CH₃)₂O— H glyasperin M OH H OH H OH —CH═CHC(CH₃)₂O— Hlicoisoflavanone H H —OC(CH₃)₂CH═CH— OH H OH H RL-Q H H —OC(CH₃)₂CH═CH—OH H OCH₃ H RL-R OH prenyl OH H OH prenyl OH H No Name

The isoflavon derivative that is a prenylflavonoid used in the presentinvention is not particularly limited, but preferable examples thereofinclude compounds represented by the following general formula (5). Forexample, compounds shown in Table 5 such as wighteone, gancaonin A,gancaonin B, gancaonin G and gancaonin N. Derivatives thereof such assalts, glycosides and esterified substances may also be mentioned. Amongthese, glyurallin B, lupiwighteone, semilicoisoflavone B andglycyrrhisoflavone are more preferable.

In the formula, at least one of R1 to R9 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 5 R1 R2 R3 R4 R5 R6 R7 R8 R9 Isoflavone(s) OH prenyl OH H H H OH HH wighteone OH prenyl OH H H H OCH₃ H H gancaonin A OH prenyl OH H H OHOCH₃ H H gancaonin B OH prenyl OCH₃ H H H OH H H gancaonin G OH prenylOH H OH H OCH₃ H H gancaonin N OH prenyl OCH₃ H OH H OH H H7-O-methylluteone OH prenyl OCH₃ H OH prenyl OH H H kanzonol K OH prenylOH prenyl —OC(CH₃)₂CH═CH— OH H H kanzonol L OH prenyl OH H H OH OHprenyl H isoangustone A OH prenyl OH H H OH —OC(CH₃)₂CH═CH— H gancaoninH OH H OH prenyl H H OH H H lupiwighteone OH H OH prenyl H OH OH H Hgancaonin L OH H OH prenyl H H OCH₃ H H gancaonin M OH H OH prenyl H OHOH prenyl H glyurallin B OH H OH prenyl —OC(CH₃)₂CH═CH— OH H Hglyasperin N H H OH H OH prenyl OH H H eurycarpin A OH H OH H OH prenylOH H H licoisoflavone A OH H OH H OH —CH═CHC(CH₃)₂O— H H licoisoflavoneB H H OH H OH —CH═CHC(CH₃)₂O— H H glabrone OH H OH H H OH OH prenyl Hglycyrrhisoflavone OCH₃ H OH H H OH OH prenyl H glisoflavone OH H OH H HOH —OC(CH₃)₂CH═CH— H semilicoisoflavone B H H OH H OH H OH prenyl OCH₃glicoricone H H OH H OH H OCH₃ prenyl OCH₃ licoricone

The flavonol derivative that is a prenylflavonoid used in the presentinvention is not particularly limited, but preferable examples thereofinclude compounds represented by the following general formula (6). Forexample, compounds shown in Table 6 such as licoflavonol, gancaonin P,topazolin, glyasperin A and isolicoflavonol. Derivatives thereof such assalts, glycosides and esterified substances may also be mentioned. Amongthese, isolicoflavonol, licoflavonol and topazolin are more preferable.

In the formula, R1 is OH or OCH₃, at least one of R2 to R9 represents aprenyl group or a structure wherein adjacent 2 R groups form a6-membered ring comprising —CH═CHC(CH₃)₂O—, and other R groups representH, OH or OCH₃.

TABLE 6 R1 R2 R3 R4 R5 R6 R7 R8 R9 Flavonol(s) OH OH prenyl OH H H H OHH licoflavonol OH OH prenyl OH H H OH OH H gancaonin P OCH₃ OH prenyl OHH H H OH H topazolin OH OH prenyl OH H H prenyl OH H glyasperin A OH OHH OH H H prenyl OH H isolicoflavonol OH OH H OH prenyl H H H Hglepidotin A OH H OH OH H prenyl OH OH H neouralenol OH OH H OH H H OHOH prenyl uralenol OCH₃ OH H OH H H OH OH prenyl uralenol-3-O-methyl-ether OCH₃ OH OH H H H OH OH prenyl uralane

The flavanone derivative that is a prenylflavonoid used in the presentinvention is not particularly limited, but preferable examples thereofinclude compounds represented by the following general formula (7). Forexample, compounds shown in Table 7 such as 6-prenylpinocembrin,6-prenylnaringenin, 6-prenyleriodictyol, sinoflavanone B andparatocarpin L. Derivatives thereof such as salts, glycosides andesterified substances may also be mentioned. Among these, glabrol ismore preferable.

In the formula, at least one of R1 to R8 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 7 R1 R2 R3 R4 R5 R6 R7 R8 Flavanone(s) OH prenyl OH H H H H H6-prenylpinocembrin OH prenyl OH H H H OH H 6-prenylnaringenin OH prenylOH H H OH OH H 6-prenyleriodictyol OH prenyl OH prenyl H H H Hsinoflavanone B OH prenyl OH H H prenyl OH H paratocarpin L H H OHprenyl H H H H ovaliflavanone B H H OH prenyl H H OH H isobavachin OH HOH prenyl H H H H glabranin OH H OH prenyl H H OH H sophoraflavanone BOH H OH prenyl H OH OH H 8-prenyleriodictyol OH H OH prenyl H OCH₃ OH Hexiguaflavanone K H H OH prenyl H prenyl OH H glabrol OH H OH prenyl Hprenyl OH H euchrestaflavanone A OH H OH prenyl H OH OH prenyl gancaoninE OH H —OC(CH₃)₂CH═CH— H H OH H citflavanone H H —OC(CH₃)₂CH═CH— Hprenyl OH H shinflavanone H H —OC(CH₃)₂CH═CH— H —CH═CHC(CH₃)₂O— Hxambioona OH H OH H H prenyl OH H licoflavanone OH H OH H H OH OH prenylsigmoidin B (uralenin) OH H OH H H H —OC(CH₃)₂CH═CH— sigmoidin C Hprenyl OH prenyl H H OH H No Name

The chalcone derivative that is a prenylflavonoid used in the presentinvention is not particularly limited, but preferable examples thereofinclude compounds represented by the following general formula (8). Forexample, compounds shown in Table 8 such as licochalcone C, licochalconeD, glyinflanin G, kanzonol B and kanzonol C. Derivatives thereof such assalts, glycosides and esterified substances may also be mentioned. Amongthese, licochalcone C is more preferable.

In the formula, at least one of R1 to R9 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 8 R1 R2 R3 R4 R5 R6 R7 R8 R9 Chalcone(s) OCH₃ prenyl OH H H H OH HH licochalcone C OCH₃ OH OH H H prenyl OH H H licochalcone D H—CH═CHC(CH₃)₂O— OH OH —CH═CHC(CH₃)₂O— H H glyinflanin G H—CH═CHC(CH₃)₂O— H OH H OH H H kanzonol B H prenyl OH H H H OH prenyl OHkanzonol C H H OH H OH H OCH₃ prenyl H isobavachalcone H prenyl OH H OHprenyl OH H H No Name H OH —OC(CH₃)₂CH═CH— OH prenyl OH H H No Name H—CH═CHC(CH₃)₂O— OH H H OH prenyl OH No Name

The dibenzoylmethane derivative that is a prenylflavonoid used in thepresent invention is not particularly limited, but preferable examplesthereof include compounds represented by the following general formula(9). For example, compounds shown in Table 9 such as glycyrdione A,glycyrdione B, glycyrdione C, glyinflanin B and glyinflanin D.Derivatives thereof such as salts, glycosides and esterified substancesmay also be mentioned. Among these, glycyrdione A and glycyrdione C aremore preferable.

In the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.

TABLE 9 R1 R2 R3 R4 R5 R6 R7 Dibenzoylmethane(s) OH H OH prenyl H prenylOH glycyrdione A (glyinflanin A) OH H OH prenyl H —CH═CHC(CH₃)₂O—glycyrdione B OH H —OC(CH₃)₂CH═CH— H prenyl OH glycyrdione C(glyinflanin C) OH H —OC(CH₃)₂CH═CH— H H OH glyinflanin B OH H—OC(CH₃)₂CH═CH— H —CH═CHC(CH₃)₂O— glyinflanin D OH H OH H H prenyl OHkanzonol A OH H OH prenyl H H OH 5′-prenyl-licodione

The chalcone derivative exclusive of prenylflavonoids used in thepresent invention is not particularly limited, but preferable examplesthereof include echinatin, isoliquiritigenin, licochalcone A andlicochalcone B. Derivatives thereof such as salts, glycosides andesterified substances may also be mentioned.

The flavonol derivative exclusive of prenylflavonoids used in thepresent invention is not particularly limited, but preferable examplesthereof include kaempferol 3-O-methylester. Derivatives thereof such assalts, glycosides and esterified substances may also be mentioned.

For names of these compounds, a report of T. Hiraga & K. Kajiyama (MeijiPharmaceutical University Bulletin, 27, 9-57, 1997) and a report of T.Nomura & T. Fukai (Fortschritte der Chemie organischer Naturstoffe, 73,1-158, 1998) were referred to.

The PPAR ligand of the present invention may be any compound derivedfrom natural sources such as plants, chemically synthesized orbiosynthesized in cultured cells, etc., if the compound is the abovecompound, but is preferably a compound derived from natural sourceswhich human can eat. The method for obtaining these compounds is notparticularly limited, and the compounds can be obtained from licorice ofthe Leguminosae family which human can eat, or from other plants. Theother plants mentioned above are not particularly limited provided thatthe plants contain the PPAR ligand, preferably the PPARγ ligand, and inaddition to the plants of the Leguminosae family, and plants of theMoraceae family, Eucommiaceae family, Cannabaceae family and Urticaceaefamily can be mentioned other than Leguminosae family.

Hereinafter, the process for producing the PPAR ligand of the presentinvention from licorice is described as an example, but the startingmaterial is not necessarily limited to licorice.

The usable licorice may be a plant of the genus Glycyrrhiza in theLeguminosae family, and examples thereof include Glycyrrhiza uralensisFisch. et DC, G. inflata BAT., G. glabra L., G. glabra L. var glandurifera Regel et Herder, G. echinata L., G. pallidiflora Maxim, and otherplants of the same genus (Leguminosae). These plants are grown in, forexample, Xin Jiang, Northeast China, Northwest China, Mongol, Russia,Afghanistan, Iran, Turkey, etc. Among them, those having a highercontent of the PPAR ligand active ingredient, such as G. uralensis(growing areas: Northeast China, Northwest China, Mongol, Xin Jiang,etc.), G. glabra (growing areas: Russia, Afghanistan, Iran, Turkey,etc.) and G. inflata (growing areas: Xin Jiang, etc.) are preferable,and G. uralensis is particularly preferably used.

In the present invention, a root, a rhizome or a stolon of the licoricementioned above is preferably used, and it is used in the form of afinely ground product, a ground product, a cut product or the peridermof the above-mentioned licorice. The finely ground product refers to apowdery or almost powdery product, and the ground product refers to aproduct in the form of filament, fiber or cotton (e.g. a product havinga length of about 5 mm to about 10 cm). The cut product is one obtainedby cutting a licorice root, a rhizome or a stolon (e.g. a thin roundslice having a length of usually about 1 to 10 cm, preferably about 1 to5 cm, more preferably about 2 to 4 cm, and a thickness of usually about3 cm or less, preferably about 2 cm or less, more preferably about 1 cmor less) usually in a cylindrical form or a substantially cylindricalform. The finely ground product, ground product and cut product can beobtained by using a conventional apparatus. The finely ground productcan be obtained as a powdery or almost powdery product by aMasscolloider type mill, grinding mortar, etc., and the ground productcan be obtained by a grinding hammer mill or the like. The cut productcan be obtained by cutting a root or the like of the licorice intopieces to have the above-mentioned length with a conventional cuttingmachine.

The composition and content of the active ingredient of the licoricevary to some degree depending on the species, growing area, harvesttime, etc. thereof. Therefore, it is preferable to use one confirmed tocontain a large amount of the active ingredient by a preliminaryexperiment.

The extraction methods include methods which comprise extracting thelicorice with an organic solvent. But a water extraction residueobtained by extracting the licorice with water, or a dried product ofthe residue, may also be subjected to extraction with an organicsolvent.

The organic solvent used herein is not particularly limited, but ispreferably a safe one usable in producing and processing ofpharmaceutical preparations, foods, food additives, etc. Examplesthereof include acetone, a monohydric alcohol having 1 to 4 carbonatoms, glycerin, fatty acid esters, diethyl ether, cyclohexane, carbondioxide, propylene glycol, etc., and two or more of these solvents maybe used as a mixture. Solvents preferably used, among these, are fattyacid esters such as ethyl acetate, or water-soluble organic solventssuch as a monohydric alcohol having 1 to 4 carbon atoms, acetone,propylene glycol, glycerin, etc., and particularly preferably thewater-soluble organic solvents.

After extraction, it is preferable to increase the content of the activeingredient in the extract by purification such as adsorption treatmentwith an activated carbon, a resin or the like and/or fractionation. Thematerial obtained by extraction may be subjected to columnchromatography on a silica gel, ODS, an ion-exchange resin, etc.,whereby the objective compound can be concentrated, fractionated orisolated.

As a matter of course, the objective compound can also be obtained byother chemical syntheses.

Hereinafter, a method of extracting licorice with a fatty acid ester ora water-soluble organic solvent is described in more detail as apreferable method of producing a licorice extract containing the PPARligand according to the present invention. The licorice extractextracted with a fatty acid ester or a water-soluble organic solventtends to exhibit better PPAR ligand activity, and the water-solubleorganic solvent is particularly preferably used because the PPAR ligandactive substance can be efficiently obtained.

When the objective substance is extracted from the licorice with a fattyacid ester or a water-soluble organic solvent, a finely ground product,ground product, cut product or periderm of licorice may be extracted asit is, but as described above, a pre-extracted licorice residue obtainedby preliminarily extracting from licorice with another solvent (e.g.water, an aqueous alkaline solution, etc.) may be extracted with a fattyacid ester or a water-soluble organic solvent to remove impurities, etc.

After pre-extraction from licorice with another solvent (water, anaqueous alkaline solution, etc.), the preliminary extract may beseparated into an extract and an extracted licorice residue by a generalseparation procedure (e.g. filtration under pressure, vacuum filtration,filter press, centrifugation, sedimentation, etc.) to give apreliminarily extracted licorice residue. In this separation procedure,generally usable filter aids and/or adsorbents such as activated carbonand activated clay, etc. can be used if necessary.

The preliminarily extracted residue thus obtained is used in extractionwith a fatty acid ester or a water-soluble organic solvent as such orafter removal of a part or the whole of another solvent used (water, anaqueous alkaline solution, etc.) by a general drying procedure (e.g.drying in a stationary state, drying under stirring, mixing drying,drying in a fluidized state, flash drying, spray drying, freeze drying,freeze concentration, etc.).

The fatty acid ester or water-soluble organic solvent which can bepreferably used in this extraction method includes, but is not limitedto, acetates such as ethyl acetate, monohydric alcohols having 1 to 4carbon atoms, acetone, propylene glycol, glycerin and mixtures thereof.Preferred among them are acetates such as ethyl acetate, monohydricalcohols having 1 to 4 carbon atoms, acetone or a mixture thereof, andmore preferred are ethyl acetate, ethanol, acetone or mixtures thereof.In particular, ethanol is preferred in view of safety of the solvent toa human body. For reducing contamination with water-soluble impuritiessuch as glycyrrhizin described later, a monohydric alcohol having 2 to 4carbon atoms, fatty acid ester and acetone are preferred, fatty acidester and acetone are particularly preferred, acetates and acetone arefurthermore preferred, and ethyl acetate and acetone are still morepreferred. Needless to say, other solvents may coexist in such a rangeas to be free of adverse effect.

In the present invention, it is important to increase the content of theactive ingredient in a licorice extract, while reducing the content ofwater-soluble impurities such as glycyrrhizin.

Glycyrrhizin is also utilized as a pharmaceutical preparation, but hasside effects such as increasing blood pressure, cardiovascular disordersand hydraemia (these three refer to Harders, H. & Rausch-Stroomann, J.G., Munch. Med. Wschr. 95, 580 (1953)), hypokalemia, a reduction inplasma renin activity, pseudo-aldosterone symptoms andextremities-relaxing paralysis (these four refer to Conn, J. W., Rovner,D. R. & Cohen, E. L., J. Am. Med. Assoc. 205, 492 (1968)), reducedsecretion of aldosterone into urine, edema, headache and drowsiness(these four refer to Epstein, M. T., et al., Brit. Med. J., 1, 488(1977)). And glycyrrhizin is 150 times as sweet as sucrose.Contamination with glycyrrhizin inhibits various effects of a licoriceextract and causes side effects and strong sweetness, thus bringingabout disadvantages in application to foods, etc.

For increasing the content of the active ingredient in the resultinglicorice extract and reducing the content of water-soluble impuritiessuch as glycyrrhizin at the same time, the content of water coexistingat the time of extraction with an organic solvent is preferably reduced.For reducing the content of water, it is important to use a licoricedried to the maximum degree, and also to use a water-free organicsolvent.

However, needless to say, licorice is a plant, and conventional sundrying is not always enough to obtain sufficiently dried licorice, thusit becomes necessary to use a drying machine, etc. This is a seriousweak point in production on a commercial scale.

Even if a water-free organic solvent is used, it is difficult tocompletely prevent the organic solvent from being contaminated withwater from the licorice used and working atmosphere, and is thusrecovered as an organic solvent increased in water content. Because theorganic solvent is hardly recycled as it is, the solvent is discarded orspecial and expensive purification facilities for making it water-freeare necessary, thus leading to an increase in production costs.

Accordingly, if a high-quality licorice extract with a higher content ofthe active ingredient and a lower content of water-soluble impuritiescan be produced quite easily and inexpensively, it is possible to expectsignificant advantages.

As a result of intensive study, the present inventors found that it ispreferable to use licorice in such a form that the ratio of the skinarea to the whole surface area thereof is high, usually 30% or more,preferably 50% or more, more preferably 70% or more, still morepreferably 80% or more, further more preferably 90% or more(specifically, the cut product or the licorice periderm (including aproduct mainly comprising periderm)), in order to obtain a high-qualitylicorice extract. Furthermore, the inventors found that when thelicorice in the above form is used, a high-quality licorice extract canbe obtained even if a hydrated water-soluble organic solvent is used.

Between the cut product and the periderm (including a product mainlycomprising periderm), the cut product is more preferably used since itcan be processed more easily, and the ratio of the skin area to thewhole surface area of the cut product is usually 50% or more, preferably70% or more, more preferably 80% or more, and still more preferably 90%or more.

According to this method, the effect can be maximized when usingparticularly the above-mentioned water-soluble organic solvent,preferably a monohydric alcohol having 2 to 4 carbon atoms, morepreferably ethanol.

The water content of the organic solvent used is usually about 30% byvolume or less, preferably about 20% by volume or less, more preferablyabout 10% by volume or less, and still more preferably about 8% byvolume or less. The lower limit is not particularly limited, but isusually about 3% by volume, preferably 4% by volume, from a practicalpoint of view.

Thereby, a licorice extract having a higher content of the activeingredient and a lower content of water-soluble impurities can bepreferably obtained. The content of glycyrrhizin in the resultingextract can be minimized preferably to 0.5% by weight or less. Even whenan inexpensive hydrated water-soluble organic solvent is used as theextraction solvent, a high-quality extract can be preferably obtained(without specially drying the licorice).

Extraction with the fatty acid ester or the water-soluble organicsolvent mentioned above can be carried out according to a generalmethod, and is not particularly limited. The extraction temperature isnot particularly limited, and extraction can be carried out preferablybetween the solidification temperature and the boiling point in thesystem, generally −20 to 100° C., usually 1 to 80° C., preferably 20 to60° C.

The extraction procedure may be carried out, for example, for 0.1 houror more, preferably 0.2 hour or more, more preferably 0.5 hour or moreby using the fatty acid ester or the water-soluble organic solvent in,for example, 1- to 20-fold excess, preferably 2- to 10-fold excess,based on the volume of the licorice or the preliminarily extractedresidue. Usually, the time for each extraction is preferably about 1 to10 hours. The upper limit is not particularly limited and is about 1day, but the extraction may be carried out for a longer time. Extractionmay be carried out once or several times if necessary, and a suitablycombined mixed solvent may be used. The pressure at the time ofextraction is not particularly limited. Extraction is carried out atnormal pressures or under pressure (one to several atmosphericpressure), but may be carried out under reduced pressure if desired. Theextraction may be carried out under reflux or in a slightly pressurizedstate.

After the extraction, the preliminary extract can be separated into anextract and an extracted licorice residue by a general separationprocedure (e.g. filtration under pressure, vacuum filtration,centrifugation, sedimentation, etc.) and if necessary washed with thesolvent, to give a licorice extract. In the separation procedure,generally usable filter aids and/or adsorbents such as activated carbon,activated clay and resins can be used if necessary.

From the licorice extract thus obtained, the used solvent can be removedby a general procedure for solvent removal (e.g. concentration at normalpressure, vacuum concentration, spray drying, freeze drying, freezeconcentration, etc.), whereby a solvent-free licorice extract can beobtained. After the extraction, the extract is more preferably purifiedby adsorption treatment with an activated carbon or a resin and/orfractionation thereby increasing the content of the active ingredient inthe extract, as described above.

In the process of the present invention, a series of the aboveprocedures, particularly extraction with a solvent, or extraction with asolvent and subsequent procedures (separation of an extract, removal ofthe solvent, adsorption treatment with an activated carbon, a resin orthe like and/or fractionation, etc.), are preferably carried out under adeoxidized atmosphere such as an inert gas atmosphere using a nitrogengas, etc. For the purpose of preventing oxidation, the process can becarried out in the concomitantly presence of antioxidants such asascorbic acid, sodium ascorbate, ascorbyl palmitate, ascorbyl stearate,tocopherol, etc.

The licorice extract obtained after removal of a solvent is generallybrown (e.g. yellowish brown to dark brown).

The weight ratio (dry weight basis) of the dry licorice extract obtainedby the process to licorice as such is usually about 0.01 or more.

The content of glycyrrhizin in the extract of the present invention, ona dry weight basis, is as low as usually 0.5% by weight or less,preferably 0.3% by weight or less, more preferably 0.2% by weight orless, and still more preferably 0.1% by weight or less. When the contentof glycyrrhizin in the extract of the present invention is 0.001 to 0.5%by weight on a dry weight basis, there is virtually not a problem in theside effects and disadvantages for use.

The content of glycyrrhizin in the extract can be determined by, forexample, analysis with HPLC using commercial glycyrrhizin orglycyrrhizinate as a standard substance or the like analysis. Thecontent of a PPAR ligand in the extract can be determined by, forexample, analysis with HPLC using the PPAR ligand of the presentinvention as a standard substance or the like analysis. The PPAR ligandas a standard substance may be a compound isolated from a plant such aslicorice or a chemically synthesized compound. The total amount of PPARligand components in the extract can be calculated as the total contentof the quantified PPAR ligand components.

The total amount of the PPAR ligand components contained in the extractof the present invention, on a dry weight basis, is usually 0.5% byweight or more, preferably 1% by weight or more, and more preferably 2%by weight or more. In particular, when the PPAR ligand is extracted witha monohydric alcohol having 1 to 4 carbon atoms, its amount is usually5% by weight or more, preferably 6% by weight or more, more preferably7% by weight or more, thus indicating efficient recovery of the activeingredient. The total amount of the PPAR ligand components contained inthe extract of the present invention, on a dry weight basis, is usuallynot higher than 50% by weight at the maximum, but the content can befurther increased by adsorption treatment with an activated carbon, aresin or the like and/or fractionation.

For example, the extract of the present invention preferably comprises,for example, at least one species selected from the group consisting ofglycycoumarin, glycyrin, dehydroglyasperin C, dehydroglyasperin D,glyasperin B and glyasperin D as a major component in an amount ofusually 0.5% by weight or more, preferably 1% by weight or more, on adry weight basis. More preferably, the extract comprises glycycoumarin,glycyrin, dehydroglyasperin C, dehydroglyasperin D, glyasperin B andglyasperin D respectively in an amount of usually 0.5% by weight ormore, preferably 1% by weight or more, on a dry weight basis. Thesecomponents are contained in a larger amount in an extract of licorice,particularly G. uralensis.

Additionally, the extract of the present invention preferably comprises,for example, at least one species selected from the group consisting ofglycyrrhisoflavanone, glycyrrhisoflavone, glyurallin B,semilicoisoflavone B and isoliquiritigenin in an amount of usually 0.01%by weight or more, preferably 0.02% by weight or more, on a dry weightbasis. More preferably, the extract comprises glycyrrhisoflavanone,glycyrrhisoflavone, glyurallin B, semilicoisoflavone B andisoliquiritigenin respectively in an amount of usually 0.01% by weightor more, preferably 0.02% by weight or more, on a dry weight basis.

Moreover, the extract of the present invention preferably comprises, forexample, at least one species selected from the group consisting ofglabrene, glabridin, glabrol, 3′-hydroxy-4′-O-methylglabridin,4′-O-methylglabridin and hispaglabridin B in an amount of usually 0.5%by weight or more, preferably 1% by weight or more, on a dry weightbasis. More preferably, the extract comprises glabrene, glabridin,glabrol, 3′-hydroxy-4′-O-methylglabridin, 4′-O-methylglabridin andhispaglabridin B respectively in an amount of usually 0.5% by weight ormore, preferably 1% by weight or more, on a dry weight basis. Thesecomponents are contained in a larger amount in an extract of licorice,particularly G. glabra.

Furthermore, the extract of the present invention preferably comprises,for example, at least one species selected from the group consisting oflicochalcone A, licochalcone B, licochalcone C, glycyrdione A andglycyrdione C in an amount of usually 0.5% by weight or more, preferably1% by weight or more, on a dry weight basis. More preferably, theextract more preferably comprises licochalcone A, licochalcone B,licochalcone C, glycyrdione A and glycyrdione C respectively in anamount of usually 0.5% by weight or more, preferably 1% by weight ormore, on a dry weight basis. These components are contained in a largeramount in an extract of licorice, particularly G. inflata.

The composition for amelioration of insulin resistance or for preventionand/or amelioration of the insulin resistance syndrome of the presentinvention, and the composition for prevention and/or amelioration ofinflammations or cancers of the present invention are compositionscomprising the PPAR ligand of the present invention. That is, they arecompositions which comprise at least one compound selected from thegroup consisting of a prenylflavonoid, a chalcone derivative exclusiveof prenylflavonoids, a flavonol derivative exclusive ofprenylflavonoids, and a salt, a glycoside and an esterified substancethereof acceptable as a pharmaceutical preparation or a food or abeverage.

As described above, the prenylflavonoid is not particularly limited, butis preferably at least one compound selected from the group consistingof a 3-arylcoumarin derivative, an isoflav-3-ene derivative, anisoflavan derivative, an isoflavanone derivative, an isoflavonederivative, a flavonol derivative, a flavanone derivative, a chalconederivative and a dibenzoylmethane derivative.

The above compound may be a pure compound or a semi-purified or crudecompound provided that it does not contain impurities unsuitable forpharmaceutical preparations and foods. In this case, the extract may beused as it is, or may be further purified. The compound is not limitedin its form and can be used as, for example, foods or beverages such ashealth-promoting foods (specified healthful foods, nutrition functionalfoods), health foods and nutrition supplementary foods, pharmaceuticalpreparations, and quasi drugs.

The composition of the present invention can be administered to human,and also to domestic animals or pets such as dogs, cats, bovine species,equine species, swine species, chickens, sheep, goats, mice, and rats.For use as a food or a beverage, it can be directly ingested or may beformulated into easily ingestable products, such as capsules, tablets,granules, powders, etc., with the aid of a known carrier, an auxiliaryagent or the like for ingestion. Furthermore, material for preparationother than the PPAR ligand of the present invention can be suitablyadded and mixed by a conventional method. Such material is notparticularly restricted, and there may be mentioned, for example,excipients, disintegrators, lubricants, binders, antioxidants, coloringagents, aggregation inhibitors, absorption promoters, stabilizers, etc.The amount of the PPAR ligand of the present invention in such aformulated product may be 0.1 to 100% by weight preferably 10 to 90% byweight. Furthermore, it can be mixed into raw materials for all kinds offood or beverage products, for example, confections such as chewing gum,chocolate, candies, jellies, biscuits and crackers; frozen sweets suchas ice cream and ice candies; beverages such as tea, nonalcoholicbeverages, nutritional drinks, and drinks for beauty; noodles such asJapanese wheat noodles, Chinese noodles, spaghetti, and instant noodles;fish paste foods such as fish minced and steamed (kamaboko), fishsausage (chikuwa), and minced flesh (hannpen); seasonings such asdressings, mayonnaise and sauces; oleaginous products such as margarine,butter and salad oil; bakery products, hams, soups, retort foods, frozenfoods, and so forth. In taking such a food or beverage composition, therecommended daily intake for an adult human is 0.1 to 3000 mg/kg, morepreferably 1 to 300 mg/kg, as the PPAR ligand of the present invention.Such compositions can also be used as feeds for domestic animals andpets or as pet foods, and the recommended daily intake in theseapplications is preferably 0.1 to 3000 mg/kg as the PPAR ligand of thepresent invention.

For use as a pharmaceutical product, the dosage form is not particularlyrestricted but includes capsules, tablets, granules, powders,injections, suppositories, patches, etc. Such dosage forms can beprepared by suitably formulating pharmaceutically acceptable materialfor preparation such as excipients, disintegrators, lubricants, binders,antioxidants, coloring agents, aggregation inhibitors, absorptionpromoters, solubilizing agents and stabilizers. The daily dosage of sucha preparation for adult human is 0.1 to 3000 mg/kg, preferably 1 to 300mg/kg, as the PPAR ligand of the present invention, which dosage is tobe administered once a day or in a few divided doses a day. Thecomposition can also be used as a pharmaceutical product for domesticand pet animals and the daily dosage for this application is preferably0.1 to 3000 mg/kg as the PPAR ligand of the present invention.

When the composition of the present invention is in the form of tablets,capsules or powders and when the above extract is contained as the PPARligand, the composition preferably comprises the extract in an amount of0.1 to 1000 mg per tablet.

The PPAR ligand of the present invention is a plant-derived extractcomponent or its related compound, and is considered to be low toxic.The extract obtained by the present invention can also be added to foodsto which sweetness or the like is an obstacle. It is highly stable ascompared with highly unsaturated fatty acids which are conventionallyreported PPARγ ligands, and is superior in respect of a form suitablefor foods and pharmaceutical compositions.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in more detail byreference to the Examples, but the scope of the present invention is notlimited to these Examples.

EXAMPLE 1 Extraction and Isolation of a Compound From Licorice (1)

A finely ground product (1.2 kg) of licorice (G. uralensis) was immersedin 5.5 L of ethyl acetate and extracted for 7 days at room temperaturewith light-shielded. Then, the mixture was filtered to obtain anextract. The extract was concentrated under reduced pressure to removethe solvent, and 74.0 g of an extract was obtained. The extract obtainedwas subjected to silica gel column chromatography (1500 ml) to obtain afraction eluted with chloroform:methanol=19:1 (v/v). The eluted fractionwas concentrated under reduced pressure to remove the solvent, and 55.4g of coarse fraction was obtained. This coarse fraction was purified byrepeating silica gel column chromatography, ODS silica gel columnchromatography, high performance liquid chromatography equipped with anODS column, gel filtration column chromatography, and fractional thinlayer chromatography. Thereby, Compound 1 (225 mg), Compound 2 (80.7mg), Compound 3 (19.6 mg), Compound 4 (22.2 mg), Compound 5 (3.9 mg),Compound 6 (72.8 mg), Compound 7 (28.3 mg), Compound 8 (12.1 mg),Compound 9 (6.8 mg), Compound 10 (8.5 mg), Compound 11 (58.7 mg) andCompound 12 (10.2 mg) were obtained.

As the result of structural analysis, Compounds 1 to 3 and 5 to 12 areknown compounds, and respectively identified as follows; Compound 1 wasglycycoumarin, Compound 2 was glycyrin, Compound 3 was glyurallin B,Compound 5 was echinatin, Compound 6 was isolicoflavonol, Compound 7 wasdehydroglyasperin C, Compound 8 was glyasperin B, Compound 9 wasglycyrrhisoflavanone, Compound 10 was lupiwighteone, Compound 11 wasglyasperin D, and Compound 12 was semilicoisoflavone B. Additionally, inthe structural identification of these compounds, spectrum datadescribed in S. Demizu, et al., Chemical and Pharmaceutical Bulletin,36, 3474-3479 (1988) was referred to for Compound 1, T. Kinoshita, etal., Chemical and Pharmaceutical Bulletin, 26, 135-140 (1978) wasreferred to for Compound 2, M. Shibano, et al., Heterocycles, 45,2053-2060 (1997) was referred to for Compounds 3 and 7, K. Kajiyama, etal., Phytochemistry, 31, 3229-3232 (1992) was referred to for Compound5, T. Hatano, et al., Chemical and Pharmaceutical Bulletin, 36,2090-2097 (1988) was referred to for Compounds 6 and 9, L. Zeng, et al.,Heterocycles, 34, 575-587 (1992) was referred to for Compounds 8 and 11,Y. Hashidoko, et al., Agricultural and Biological Chemistry, 50,1797-1807 (1986) was referred to for Compound 10, and F. Kiuchi, et al.,Heterocycles, 31, 629-636 (1990) was referred to for Compound 12.

Compound 4 is a novel compound, and its structure was determined as3-(2′,4′-dihydroxyphenyl)-6-(3″,3″-dimethylallyl)-5,7-dimethoxy-2H-chromeneby a spectrum analysis centered on dimensional NMR (¹H-²H COSY, HMQC,HMBC, PHNOESY), and named dehydroglyasperin D.

The characteristic and spectrum data of Compound 4 are as follows.

Dehydroglyasperin D, brown powder, C₂₂H₂₄O₅.

EI-MS m/z: 368.1610[M]+(calculated value; C₂₂H₂₄O₅: 368.1624).

Uvλmax (methanol) nm: 330 (log ε=4.25), 243 sh (log ε=4.21).

IR (KBr tablet) cm⁻¹: 3375, 2929, 1613, 1516, 1458, 1308, 1198, 1164,1114, 1092, 1024, 978, 837.

¹H-NMR (dimethyl disulfoxide) ppm: 7.06 (1H, d, J=8.4 Hz, H-6′), 6.68(1H, s, H-4), 6.34 (1H, d, J=2.3 Hz, H-3′), 6.33 (1H, s, H-8), 6.26 (1H,dd, J=8.4, 2.3 Hz, H-5′), 5.09 (1H, br t, J=6.9 Hz, H-10), 4.90 (2H, s,H-2), 3.75 (3H, s, C-7-OMe), 3.67 (3H, s, C-5-OMe), 3.18 (2H, br d,J=6.7 Hz, H-9), 1.71 (3H, s, Me-13), 1.63 (3H, s, Me-12).

¹³C-NMR (dimethyl disulfoxide) ppm: 67.9 (C-2), 128.8 (C-3), 114.7(C-4), 110.4 (C-4a), 154.9 (C-5), 115.6 (C-6), 158.0 (C-7), 95.8 (C-8),153.2 (C-8a), 22.6 (C-9), 124.1 (C-10), 130.4 (C-11), 26.0 (C-12), 18.1(C-13), 116.8 (C-1′), 156.7 (C-2′), 103.3 (C-3′), 158.7 (C-4′), 107.4(C-5′), 129.2 (C-6′), 62.3 (C-5-OMe), 56.3 (C-7-OMe).

EXAMPLE 2 Extraction and Isolation of a Compound From Licorice (2)

In the same procedure as in Example 1, Compounds 13 to 17 were obtainedby extracting a finely ground product of licorice (G. uralensis) withethanol to carry out various chromatography. As a result of structuralanalysis, the Compounds were identified as follows; Compound 13 wasisoliquiritigenin, Compound 14 was kaempferol 3-O-methyl ester, Compound15 was licoflavonol, Compound 16 was topazolin, and Compound 17 wasglycyrrisoflavone.

EXAMPLE 3 Extraction and Isolation of a Compound from Licorice (3)

In the same procedure as in Example 1 or 2, Compounds 18 to 23 andCompounds 24 to 28 were obtained by extracting a finely ground productof licorice (G. glabra) and a finely ground product of licorice (G.inflata) with ethanol, respectively. As a result of structural analysis,the Compounds were identified as follows; Compound 18 was glabridin,Compound 19 was glabrene, Compound 20 was hispaglabridin B, Compound 21was 4′-O-methylglabridin, Compound 22 was3′-hydroxy-4′-O-methylglabridin, Compound 23 was glabrol, Compound 24was licochalcone A, Compound 25 was licochalcone B, Compound 26 waslicochalcone C, Compound 27 was glycyrdione A, and Compound 28 wasglycyrdione C.

Tables 10 to 13 show the structural formulae of Compounds 1 to 28.

TABLE 10 Compound Structural number Compound name formula Compound 1Glycycoumarin

Compound 2 Glycyrin

Compound 3 Glyurallin B

Compound 4 Dehydroglyasperin D

Compound 5 Echinatin

Compound 6 Isolicoflavonol

Compound 7 Dehydroglyasperin C

Compound 8 Glyasperin B

TABLE 11 Compound Structural number Compound name formula Compound 9Glycyrrhisoflavanone

Compound 10 Lupiwighteone

Compound 11 Glyasperin D

Compound 12 Semilicoisoflavone B

Compound 13 Isoliquiritigenin

Compound 14 Kaempferol3-O-methylester

Compound 15 Licoflavonol

TABLE 12 Compound Structural number Compound name formula Compound 16Topazolin

Compound 17 Glycyrrhisoflavone

Compound 18 Glabridin

Compound 19 Glabrene

Compound 20 Hispaglabridin B

Compound 21 4′-O-Methylglabridin

Compound 22 3′-Hydroxyl-4′-O-methylglabridin

TABLE 13 Compound Structural number Compound name formula Compound 23Glabrol

Compound 24 Licochalcone A

Compound 25 Licochalcone B

Compound 26 Licochalcone C

Compound 27 Glycyrdione A

Compound 28 Glycyrdione C

EXAMPLE 4 Measurement of PPARγ Ligand Activity

CV-1 cells (cultured cells derived from the kidney of a male Africangreen monkey) were inoculated on a 96-well culture plate in aconcentration of 6×10³ cells/well and incubated at 37° C. in 5% CO₂ for24 hours. The medium used was DMEM (Dulbecco's Modified Eagle Medium:GIBCO) containing 10% FBS (fetal bovine serum), 10 ml/L penicillin andstreptomycin solution (containing 5000 IU/ml and 5,000 μg/ml,respectively: GIBCO) and 37 mg/L ascorbic acid (Wako Pure ChemicalIndustries, Ltd.) The cells were washed with OPTI-MEM (GIBCO) and thentransfected with pM-mPPARγ and 4×UASg-luc by means of Lipofect AminePlus (GIBCO). pM-mPPARγ is a plasmid for chimera protein expressioncomprising yeast-derived transcription factor GAL4 gene (amino acidsequence at 1 to 147) and a mouse PPARγ ligand-binding site gene (aminoacid sequence at 174 to 475) bound thereto. 4×UASg-luc is a reporterplasmid comprising four GAL4 response sequences (UASg) integrated into aregion upstream of a luciferase gene. At about 24 hours aftertransfection, the medium was exchanged with a sample-containing medium,and the cells were cultured for 24 hours (n=4). The sample-containingmedium was prepared by dissolving a sample in dimethyl sulfoxide (DMSO)in an amount of 1/1000 based on the medium, while for the untreatedcontrol, DMSO in an amount of 1/1000 based on the medium was used inplace of the sample. After the cells were washed with a Ca- andMg-containing phosphate buffered physiological saline (PBS+), LucLite(Packard) was added thereto, and the intensity of luciferaseluminescence was measured with a TopCount microplatescintillation/luminescence counter (Packard).

As the control group, pM (plasmid from which the PPARγ ligand-bindingsite gene was removed) was measured in place of pM-mPPARγ in the samemanner as for the measurement group. For each sample, the ratio(measurement group/control group) of the average luminescence intensityof the measurement group (n=4) to that of the control group wascalculated, and the specific activity relative to the activity of theuntreated control was regarded as the PPARγ ligand activity of thesample.

Tables 14 to 17 show the measurement results of the PPARγ ligandactivities of Compounds 1 to 28 obtained in Examples 1 to 3. Ascomparative compounds, glycyrrhizin (glycyrrhizic acid, Wako PureChemical Industries, Ltd.), glycyrrhetinic acid (Wako Pure ChemicalIndustries, Ltd.) and quercetin (Sigma) were measured for their PPARγligand activity.

TABLE 14 Added PPARγ concentration ligand activity Untreated control(DMSO) 0.1% 1.00 Troglitazone 0.5 μM   2.17 ± 0.08 1 μM 3.36 ± 0.24 2 μM4.60 ± 0.27 Compound 1 2 μg/ml (5.4 μM)  2.03 (Glycycoumarin) 5 μg/ml(13.6 μM) 2.79 10 μg/ml (27.2 μM)  3.82 Compound 2 2 μg/ml (5.2 μM) 2.81 (Glycyrin) 5 μg/ml (13.1 μM) 3.34 10 μg/ml (26.2 μM)  3.67 Compound3 2 μg/ml (4.7 μM)  1.28 (Glyurallin B) 5 μg/ml (11.8 μM) 1.96 10 μg/ml(23.7 μM)  2.73 Compound 4 2 μg/ml (5.4 μM)  2.95 (Dehydroglyasperin D)5 μg/ml (13.6 μM) 3.72 10 μg/ml (27.2 μM)  4.00 Compound 5 2 μg/ml (7.4μM)  1.45 (Echinatin) 5 μg/ml (18.5 μM) 2.35 10 μg/ml (37.0 μM)  2.49

TABLE 15 Added PPARγ concentration ligand activity Untreated control(DMSO) 0.1% 1.00 Troglitazone 0.5 μM   2.44 ± 0.10 1 μM 3.99 ± 0.24 2 μM5.74 ± 0.31 Compound 6 2 μg/ml (5.6 μM)  1.73 (Isolicoflavonol) 5 μg/ml(14.1 μM) 2.50 10 μg/ml (28.2 μM)  3.82 Compound 7 2 μg/ml (5.6 μM) 2.90 (Dehydroglyasperin C) 5 μg/ml (14.1 μM) 3.73 10 μg/ml (28.2 μM) 4.41 Compound 8 2 μg/ml (5.4 μM)  2.60 (Glyasperin B) 5 μg/ml (13.5 μM)3.53 10 μg/ml (27.0 μM)  3.02 Compound 9 2 μg/ml (5.4 μM)  2.01(Glycyrrhisoflavanone) 5 μg/ml (13.6 μM) 3.45 10 μg/ml (27.2 μM)  4.68Compound 10 2 μg/ml (5.9 μM)  1.67 (Lupiwighteone) 5 μg/ml (14.8 μM)2.53 10 μg/ml (29.6 μM)  3.75 Compound 11 2 μg/ml (5.4 μM)  3.22(Glyasperin D) 5 μg/ml (13.5 μM) 3.28 10 μg/ml (27.0 μM)  4.06 Compound12 2 μg/ml (5.7 μM)  1.86 (Semilicoisoflavone B) 5 μg/ml (14.2 μM) 2.6610 μg/ml (28.4 μM)  3.15 Glycyrrhizin 2 μg/ml (2.4 μM)  1.21 5 μg/ml(6.1 μM)  1.10 10 μg/ml (12.2 μM)  0.75 Glycyrrhetinic acid 2 μg/ml (4.2μM)  0.97 5 μg/ml (10.6 μM) 0.95 10 μg/ml (21.2 μM)  0.97 Quercetin 2μg/ml (5.9 μM)  1.15 5 μg/ml (14.8 μM) 1.72 10 μg/ml (29.6 μM)  1.70

TABLE 16 Added PPARγ concentration ligand activity Untreated control(DMSO) 0.1% 1.00 Troglitazone 0.5 μM   2.24 ± 0.56 1 μM 3.29 ± 0.60 2 μM4.80 ± 1.30 Compound 13 2 μg/ml (7.8 μM)  1.47 (Isoliquiritigenin) 5μg/ml (19.5 μM) 2.71 10 μg/ml (39.0 μM)  3.39 Compound 14 2 μg/ml (6.7μM)  1.86 (Kaempferol 3-O-methylester) 5 μg/ml (16.7 μM) 2.37 10 μg/ml(33.3 μM)  3.47 Compound 15 2 μg/ml (5.6 μM)  1.44 (Licoflavonol) 5μg/ml (14.1 μM) 2.27 10 μg/ml (28.2 μM)  4.07 Compound 16 2 μg/ml (5.4μM)  2.56 (Topazolin) 5 μg/ml (13.6 μM) 3.64 10 μg/ml (27.1 μM)  5.84Compound 17 2 μg/ml (5.6 μM)  1.78 (Glycyrrhisoflavone) 5 μg/ml (14.1μM) 3.05 10 μg/ml (28.2 μM)  3.20

TABLE 17 Added PPARγ concentration ligand activity Untreated control(DMSO) 0.1% 1.00 Troglitazone 0.5 μM   2.05 ± 0.35 1 μM 3.30 ± 0.43 2 μM5.66 ± 1.06 Compound 18 2 μg/ml (6.2 μM)  1.64 (Glabridin) 5 μg/ml (15.4μM) 2.15 10 μg/ml (30.8 μM)  1.98 Compound 19 2 μg/ml (6.2 μM)  1.78(Glabrene) 5 μg/ml (15.5 μM) 3.44 10 μg/ml (31.0 μM)  2.89 Compound 20 2μg/ml (5.1 μM)  1.04 (Hispaglabridin B) 5 μg/ml (12.8 μM) 1.45 10 μg/ml(25.6 μM)  1.94 Compound 21 2 μg/ml (5.9 μM)  1.25(4′-O-Methylglabridin) 5 μg/ml (14.8 μM) 1.55 10 μg/ml (29.6 μM)  2.13Compound 22 2 μg/ml (5.6 μM)  1.61 (3′-Hydroxyl-4′-O- 5 μg/ml (14.1 μM)2.46 methylglabridin) 10 μg/ml (28.2 μM)  1.69 Compound 23 2 μg/ml (5.1μM)  1.85 (Glabrol) 5 μg/ml (12.7 μM) 3.48 10 μg/ml (25.5 μM)  3.74Compound 24 2 μg/ml (5.9 μM)  4.02 (Licochalcone A) 5 μg/ml (14.8 μM)4.14 10 μg/ml (29.6 μM)  4.16 Compound 25 2 μg/ml (7.0 μM)  1.27(Licochalcone B) 5 μg/ml (17.5 μM) 1.93 10 μg/ml (34.9 μM)  1.61Compound 26 2 μg/ml (5.9 μM)  1.84 (Licochalcone C) 5 μg/ml (14.8 μM)2.20 10 μg/ml (29.6 μM)  3.71 Compound 27 2 μg/ml (4.9 μM)  1.44(Glycyrdione A) 5 μg/ml (12.2 μM) 2.49 10 μg/ml (24.5 μM)  3.05 Compound28 2 μg/ml (4.9 μM)  0.96 (Glycyrdione C) 5 μg/ml (12.3 μM) 1.48 10μg/ml (24.6 μM)  2.04

The above PPARγ ligand activity of each compound was compared with thatof a positive control, troglitazone (Sankyo Co., Ltd.). As a result,Compounds 1 to 19, 21 to 24, 26 and 27 were recognized to have higherPPARγ ligand activity than that of a positive control containing 0.5 μMtroglitazone. Compounds 20, 25 and 28 were also recognized to have PPARγligand activity, although it was slightly weaker than that of a positivecontrol containing 0.5 μM troglitazone. However, glycyrrhizin that is ahydrophilic component and a major component of licorice, glycyrrhetinicacid (derivative of glycyrrhizin whose sugar residue was hydrolyzed),and quercetin (flavonol derivative of Compound 6 in which OH groupsubstitutes prenyl group), were not recognized to have a PPARγ ligandactivity. The fact that quercetin does not have a PPARγ ligand activityaccords with information in a literature (Liang, Y. C., et al., FEBSLetters, 496, 12-18, 2001).

EXAMPLE 5 Effect on Type 2 Diabetes Mellitus Model Mouse

Using a KK-Ay mouse which is genetically obese and type 2 diabeticanimal, the effect of Compound 2 on diabetes mellitus was evaluated. Fora positive control, pioglitazone, i.e. an agent for ameliorating insulinresistance/agent for treating type 2 diabetes mellitus was used.

KK-Ay mice (female, 15-week-old) were divided into 3 groups eachconsisting of 5 mice, and freely given a feed comprising only a basaldiet, i.e. powdered CE-2 (CLEA Japan, Inc.), to a non-additive group(control group), a feed comprising a basal diet and pioglitazone to apioglitazone-given group, and a feed comprising a basal diet andCompound 2 to a Compound 2-given group. As for pioglitazone, ACTOSTablet 30 (containing 30 mg pioglitazone per tablet, Takeda ChemicalIndustries, Ltd.) was milled with an agate mortar and added to the basaldiet such that the content of pioglitazone in a final feed was 0.02%.Compound 2 was prepared in the same procedure as in Example 1 and addedto the basal diet such that the content of Compound 2 in a final feedwas 0.1%. On the day before administration and 4 days afteradministration, a small amount of blood was collected from a mouse tailvein, and measured for its blood glucose level by a simple blood glucosemeasuring instrument Glutest Ace (Sanwa Kagaku Kenkyusho Co., Ltd.).Table 18 shows the results.

After the mixed feed was given for 1 week, the mice were fastedovernight and then subjected to a glucose tolerance test. That is, 20mg/kg of pioglitazone, or 100 mg/kg of Compound 2, suspended in 0.5%carboxymethyl cellulose-sodium (CMC—Na) solution, was forcibly orallyadministered into the fasted mice. To the non-additive group (controlgroup), 0.5% CMC—Na solution was administered in an amount of 5 ml/kg.At a time of 30 minutes after administration, the mice were loaded with2 g/kg of 40% sucrose solution. Before the sugar loading and at a timeof 30 minutes, 1 hour and 2 hours after the sugar loading, a smallamount of blood was collected from a mouse tail vein, and measured forits blood glucose level by a simple blood glucose measuring instrumentGlutest Ace (Sanwa Kagaku Kenkyusho Co., Ltd.). Table 19 shows theresults.

TABLE 18 Non-additive Compound 2 Blood glucose group Pioglitazone-(Glycyrin)- level(mg/dl) (control group) given group given group Before476 ± 22 486 ± 26 474 ± 27 administration 4 days after 420 ± 14  191 ±6**  278 ± 14** administration **(p < 0.01)

TABLE 19 Non-additive Compound 2 Blood glucose group Pioglitazone-(Glycyrin)- level (mg/dl) (control group) given group given group Beforesugar loading 80 ± 2 89 ± 3 78 ± 4 30 minutes after sugar 355 ± 18  158± 11**  225 ± 27** loading  1 hour after sugar 199 ± 11 136 ± 5* 129 ±8* loading  2 hour after sugar 100 ± 4  106 ± 4  100 ± 3  loading *(p <0.05), **(p < 0.01

As is evident from Table 18, the blood glucose levels (means±SE, n=5) ofthe mice of Compound 2-given group were statistically significantlyreduced in a similar manner as in that of the mice of pioglitazone-givengroup, on the fourth day after the administration. As is evident fromTable 19, Compound 2, like pioglitazone, has statistically significantlyinhibited a rapid increase in blood glucose levels after the sugarloading. From these results, it was confirmed that Compound 2 has alowering action and an increase-inhibition action of blood glucoselevel, thus was suggested that the compound has an insulinresistance-ameliorating action, like pioglitazone.

EXAMPLE 6

A finely ground product (500 g) of licorice (G. uralensis) (Kaneka SunSpice Co., Ltd) was added with 5 kg of ethanol (99.5% by volume). Themixture was extracted at 25° C. for 5 hours, and the residue wasfiltered off to obtain an extract. The solvent was removed from thisextract under reduced pressure, and 45 g of a licorice extract having acolor of yellow brown to dark brown was obtained. This licorice extractexhibited good PPARγ ligand activity. Table 20 shows contents of PPARγligand active ingredients and glycyrrhizin contained in this licoriceextract.

Additionally, the contents of these active ingredients and glycyrrhizinwere obtained by the HPLC analysis under the following conditions.

(Glycyrrhizin Analysis Conditions)

Column; J′ sphere ODS-H80 (YMC Co., Ltd.) 4.6 mm(inner diameter)×250mm(length): column temperature; 40° C.: mobile phase; acetonitrile/10 mMphosphoric acid solution=33/67 (v/v): flow rate; 1 ml/min: detectionwavelength; 254 nm: retention time of glycyrrhizin; 27.1 min.

(Active Ingredient Analysis Conditions)

Column; J′ sphere ODS-H80 (YMC Co., Ltd.) 4.6 mm (inner diameter)×250 mm(length): column temperature; 40° C. Mobile phase; A gradient in whichthe ratio of acetonitrile relative to a 10 mM phosphoric acid solutionis kept to be 35% until 15 min after the start of analysis, thenincreased to 70% at a constant rate in a period from 15 min until 65min, and then kept to be 70% in a period from 65 min until 70 min. Flowrate; 1 ml/min, detection wavelength; 254 nm. Retention time for eachcomponent is as follows; 5.2 min for licochalcone B, 13.3 min forglycyrrhisoflavanone, 14.7 min for isoliquiritigenin, 32.7 min forglycycoumarin, 34.2 min for semilicoisoflavone B, 35.9 min for glabrene,36.3 min for glycyrrhisoflavone, 37.2 min for dehydroglyasperin C, 39.1min for licochalcone C, 40.2 min for licochalcone A, 44.7 min forglabridin, 48.1 min for glycyrin, 51.1 min for glyasperin B, 51.8 minfor glabrol, 52.7 min for glyasperin D, 53.4 min for glycyrdione A, 53.5min for 3′-hydroxyl-4′-O-methylglabridin, 55.2 min for dehydroglyasperinD, 58.7 min for glyurallin B, 59.8 min for 4′-O-methylglabridin, 63.0min for glycyrdione C, and 74.1 min for hispaglabridin B.

TABLE 20 Compound name Content (wt %) Glycyrrhizin 0.28Glycyrrhisoflavanone 0.15 Isoliquiritigenin 0.19 Glycycoumarin 2.55Semilicoisoflavone B 0.45 Glycyrrhisoflavone 0.50 Dehydroglyasperin C2.82 Glycyrin 1.46 Glyasperin B 1.12 Glyasperin D 2.44 DehydroglyasperinD 1.39 Glyurallin B 0.03 Active component: total 13.10%

EXAMPLE 7

A licorice extract (18 g) was obtained by the same procedure as inExample 6 except that extraction was carried out by acetone instead ofethanol. The obtained licorice extract exhibited good PPARγ ligandactivity. Contents of active ingredients and glycyrrhizin contained inthis licorice extract were examined in the same manner as in Example 6,and Table 21 shows the results.

TABLE 21 Compound name Content (wt %) Glycyrrhizin 0.06Glycyrrhisoflavanone 0.19 Isoliquiritigenin 0.16 Glycycoumarin 3.50Semilicoisoflavone B 0.01 Glycyrrhisoflavone 0.69 Dehydroglyasperin C4.09 Glycyrin 1.91 Glyasperin B 1.40 Glyasperin D 3.38 DehydroglyasperinD 1.82 Glyurallin B 0.03 Active component: total 17.18%

EXAMPLE 8

A licorice extract (18 g) was obtained by the same procedure as inExample 6 except that extraction was carried out by ethyl acetateinstead of ethanol. The obtained licorice extract exhibited good PPARγligand activity. Contents of active ingredients and glycyrrhizincontained in this licorice extract were examined in the same manner asin Example 6, and Table 22 shows the results.

TABLE 22 Compound name Content (wt %) Glycyrrhizin 0.05Glycyrrhisoflavanone 0.18 Isoliquiritigenin 0.09 Glycycoumarin 3.44Semilicoisoflavone B 0.62 Glycyrrhisoflavone 0.70 Dehydroglyasperin C4.09 Glycyrin 1.96 Glyasperin B 1.52 Glyasperin D 3.41 DehydroglyasperinD 1.93 Glyurallin B 0.03 Active component: total 17.97%

COMPARATIVE EXAMPLE 1

A licorice extract was obtained by the same procedure as in Example 6except that extraction was carried out by water instead of ethanol. Thislicorice extract exhibited no PPARγ ligand activity.

EXAMPLE 9

A finely ground product (500 g) of licorice (G. uralensis) (Kaneka SunSpice Co., Ltd) was added with 5 kg of water, and the mixture wasextracted at 60° C. for 1 day. Then, the residue was filtered off anddried under reduced pressure. The obtained pre-extracted residue wasadded with 2.5 kg of ethanol (99.5% by volume) and then extracted at 25°C. for 5 hours, and the residue was filtered off to obtain an extract.The solvent was removed from this extract under reduced pressure, and 47g of a licorice extract having a color of yellow brown to dark brown wasobtained. This licorice extract exhibited good PPARγ ligand activity.Table 23 shows contents of PPARγ ligand active ingredients andglycyrrhizin contained in this licorice extract.

TABLE 23 Compound name Content (wt %) Glycyrrhizin 0.50Glycyrrhisoflavanone 0.18 Isoliquiritigenin 1.93 Glycycoumarin 2.10Semilicoisoflavone B 0.40 Glycyrrhisoflavone 0.44 Dehydroglyasperin C1.19 Glycyrin 1.44 Glyasperin B 1.19 Glyasperin D 1.59 DehydroglyasperinD 1.03 Glyurallin B 0.03 Active component: total 11.52%

EXAMPLE 10

Licorice extracts (25.8 g and 29.5 g) were obtained by extracting cutproducts (the ratio of the total surface area of skin is about 80% ofthe total surface area of licorice) of licorice (G. uralensis) with99.5% by volume and 95.0% by volume of ethanol (water contents were 0.5and 5.0% by volume, respectively), respectively, in the same procedureas in Example 6. These licorice extracts exhibited good PPARγ ligandactivity. Table 24 shows contents of PPARγ ligand active ingredients andglycyrrhizin contained in these licorice extracts.

TABLE 24 Water content ratio of ethanol at the time of extraction 0.5vol % 5.0 vol % Compound name Content (wt %) Content (wt %) Glycyrrhizin0.02 0.26 Glycyrrhisoflavanone 0.14 0.03 Isoliquiritigenin 0.03 0.05Glycycoumarin 3.12 2.85 Semilicoisoflavone B 0.42 0.41Glycyrrhisoflavone 0.65 0.54 Dehydroglyasperin C 7.53 4.50 Glycyrin 1.951.92 Glyasperin B 3.92 3.62 Glyasperin D 6.48 4.23 Dehydroglyasperin D4.43 3.63 Glyurallin B 0.12 0.11 Active component: total 28.79% 21.89%

EXAMPLE 11

A licorice extract was obtained from a finely ground product of licorice(G. glabra) in the same procedure as in Example 6. Contents of activeingredients and glycyrrhizin contained in this licorice extract wereexamined in the same manner as in Example 6, and Table 25 shows theresults.

TABLE 25 Compound name Content (wt %) Glycyrrhizin 0.27Glycyrrhisoflavanone 0.09 Isoliquiritigenin 0.12 Glabrene 3.14 Glabridin22.08 Glabrol 4.33 3′-Hydroxyl-4′-O-methylglabridin 2.194′-O-Methylglabridin 3.03 Hispaglabridin B 1.14 Active component: total36.12%

EXAMPLE 12

A licorice extract was obtained from a finely ground product of licorice(G. inflata) in the same procedure as in Example 6. The obtainedlicorice extract exhibited good PPARγ ligand activity. Contents ofactive ingredients and glycyrrhizin contained in this licorice extractwere examined in the same manner as in Example 6, and Table 26 shows theresults.

TABLE 26 Compound name Content (wt %) Glycyrrhizin 0.17Isoliquiritigenin 0.19 Licochalcone B 2.57 Licochalcone C 3.61Licochalcone A 20.34 Glycyrdione A 1.91 Glycyrdione C 0.94 Activecomponent: total 29.56%

EXAMPLE 13

Corn starch, lactose, carboxylmethyl cellulose, and magnesium stearatewere mixed with the licorice extract obtained in Example 6. Furthermore,an aqueous solution of polyvinyl pyrrolidone was added thereto as abinder, and the mixture was granulated by a conventional method. Theresultant was added with talc and mixed, and was pressed into tabletshaving the following compositions.

Licorice extract 10 parts by weight Corn starch 25 parts by weightLactose 15 parts by weight Carboxylmethyl cellulose 10 parts by weightMagnesium stearate  3 parts by weight Polyvinyl pyrrolidone  5 parts byweight Talc 10 parts by weight

INDUSTRIAL APPLICABILITY

According to the present invention, a peroxisome proliferator-activatedreceptor (PPAR) ligand, and a composition containing thereof can beprovided easily and efficiently. The composition of the presentinvention is useful for amelioration of insulin resistance or as anagent for prevention and/or amelioration of the insulin resistancesyndrome.

1-69. (canceled)
 70. A method for preventing or ameliorating an insulinresistance syndrome which comprises administering a compositioncomprising, as an active ingredient, at least one prenylflavonoidselected from the group consisting of a 3-arylcoumarin derivative, anisoflav-3-ene derivative, an isoflavan derivative, an isoflavanonederivative, an isoflavone derivative, a flavonol derivative, a flavanonederivative, a chalcone derivative and a dibenzoylmethane derivative to asubject.
 71. The method according to claim 70, wherein theprenylflavonoid comprises a 3-arylcoumarin derivative represented by thegeneral formula (1):

in the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 72. Themethod according to claim 71, wherein the 3-arylcoumarin derivativerepresented by the general formula (1) is glycycoumarin or glycyrin. 73.The method according to claim 70, wherein the prenylflavonoid comprisesan isoflav-3-ene derivative represented by the general formula (2):

in the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 74. Themethod according to claim 73, wherein the isoflav-3-ene derivativerepresented by the general formula (2) is dehydroglyasperin D,dehydroglyasperin C or glabrene.
 75. The method according to claim 70,wherein the prenylflavonoid comprises an isoflavan derivativerepresented by the general formula (3):

in the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 76. Themethod according to claim 75, wherein the isoflavan derivativerepresented by the general formula (3) is glyasperin D, glabridin,hispaglabridin B, 4′-O-methylglabridin or3′-hydroxy-4′-O-methylglabridin.
 77. The method according to claim 70,wherein the prenylflavonoid comprises an isoflavanone derivativerepresented by the general formula (4):

in the formula, at least one of R1 to R8 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 78. Themethod according to claim 77, wherein the isoflavanone derivativerepresented by the general formula (4) is glyasperin B orglycyrrhisoflavanone.
 79. The method according to claim 70, wherein theprenylflavonoid comprises an isoflavone derivative represented by thegeneral formula (5):

in the formula, at least one of R1 to R9 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 80. Themethod according to claim 79, wherein the isoflavone derivativerepresented by the general formula (5) is glyurallin B, lupiwighteone,semilicoisoflavone B or glycyrrhisoflavone.
 81. The method according toclaim 70, wherein the prenylflavonoid comprises a flavonol derivativerepresented by the general formula (6):

in the formula, R1 is OH or OCH₃, at least one of R2 to R9 represents aprenyl group or a structure wherein adjacent 2 R groups form a6-membered ring comprising —CH═CHC(CH₃)₂O—, and other R groups representH, OH or OCH₃.
 82. The method according to claim 81, wherein theflavonol derivative represented by the general formula (6) isisolicoflavonol, licoflavonol or topazolin.
 83. The method according toclaim 70, wherein the prenylflavonoid comprises a flavanone derivativerepresented by the general formula (7):

in the formula, at least one of R1 to R8 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 84. Themethod according to claim 83, wherein the flavanone derivativerepresented by the general formula (7) is glabrol.
 85. The methodaccording to claim 70, wherein the prenylflavonoid comprises a chalconederivative represented by the general formula (8):

in the formula, at least one of R1 to R9 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 86. Themethod according to claim 85, wherein the chalcone derivativerepresented by the general formula (8) is licochalcone C.
 87. The methodaccording to claim 70, wherein the prenylflavonoid comprises adibenzoylmethane derivative represented by the general formula (9):

in the formula, at least one of R1 to R7 represents a prenyl group or astructure wherein adjacent 2 R groups form a 6-membered ring comprising—CH═CHC(CH₃)₂O—, and other R groups represent H, OH or OCH₃.
 88. Themethod according to claim 87, wherein the dibenzoylmethane derivativerepresented by the general formula (9) is glycyrdione A or glycyrdioneC.
 89. A method for preventing or ameliorating inflammations or cancerswhich comprises administering a composition comprising, as an activeingredient, at least one prenylflavonoid selected from the groupconsisting of a 3-arylcoumarin derivative, an isoflav-3-ene derivative,an isoflavan derivative, an isoflavanone derivative, an isoflavonederivative, a flavonol derivative, a flavanone derivative, a chalconederivative and a dibenzoylmethane derivative to a subject.